NDV is one of the most diverse and deadly avian pathogens. The almost simultaneous occurrence of Newcastle disease as an apparent new disease in several different geographical locations and the great variation in the severity of the disease has caused some problems with nomenclature.
The disease has been termed pseudo fowl pest, pseudo poultry plague, avian pest, avian distemper and avian pneumoencephalitis. The importance of the disease is primarily due to the development of the poultry industry during the 20th Century into a highly efficient international industry which is dependent on intensive trade between countries.
It is generally assumed that the first outbreaks of Newcastle disease occurred in 1926 in Java, Indonesia, and in Newcastle-upon-Tyne, England (Kraneveld, 1926; Doyle 1927). The name “Newcastle disease” was coined by Doyle as a temporary name to avoid a descriptive name that might be confused with other diseases. It later became clear that other less severe diseases were caused by viruses indistinguishable from NDV. In the US, a relatively mild respiratory disease was named “avian pneumoencephalitis” and was shown to be caused by NDV (Beach, 1944). Within a few years, numerous NDV isolations that caused extremely mild or no disease in chickens were made around the world.
The following methods have been implicated in the spread of the disease: 1) movement of live birds, feral birds, game birds, racing pigeons and commercial poultry; 2) movement of people and equipment; 3) movement of poultry products; 4) airborne spread; 5) contaminated poultry feed; 6) contaminated water; 7) incompletely inactivated or heterogeneous vaccines. According to the OIE, Newcastle disease is a disease of poultry caused by a virus of avian-paramyxovirus serotype 1 (APMV-1) which has an intracerebral pathogenicity index (ICPI) in day-old chicks of 017 or greater. Virulent virus can also be confirmed by the presence of multiple basic amino acids at the C-terminus of the F2 protein and F (phenylalanine) at residue 117, the N-terminus of the F1 protein. Failure to demonstrate this amino acid sequence would require characterization by ICPI tests. The word “poultry” refers to domestic fowl, turkeys, guinea fowl, ducks, geese, quails, pigeons, pheasants, partridges and ratites that are reared or kept in captivity for breeding, the production of meat or eggs for consumption, or for restocking supplies of game.
According to Alexander (1988) three panzootics of Newcastle disease have occurred since the first recognition of the disease. The first represented the initial outbreaks of the disease and appears to have arisen in Southeast Asia. Isolated outbreaks, such as the one in England in 1926, were chance introductions ahead of the mainstream which slowly moved through Asia to Europe.
A second panzootic appears to have begun in the Middle East in the late 1960's and reached most countries by 1973. The more rapid spread of the second panzootic was probably caused by the major revolution of the poultry industry with considerable international trade.
A third panzootic primarily affected domesticated birds such as pigeons and doves (Vindevogel and Duchatel, 1988). The disease apparently arose in the Middle East in the late 1970's. By 1981, it reached Europe and then spread rapidly to all parts of the world, largely as a result of contact between birds at races and shows and the international trade in such birds.
Nowadays, Newcastle disease is still widespread in many countries of Asia, Africa, the Americas, and Europe. Only the countries of Oceania appear to be relatively free from the disease (Spradbrow, 1988).
NDV belongs to the order Monomegavirales, family Paramyxoviridae, subfamily Paramyxoviridae, and genus Rubulavirus. Apart from NDV, generally called avian-paramyxovirus type-1, eight other serotypes, designated avian-paramyxovirus type-2 to -9, can be distinguished on the basis of their antigenic relatedness in hemagglutination-inhibition tests and serum neutralization tests (Alexander, 1993).
Despite the consistency of the serological grouping there are some cross-relationships between viruses of the different serotypes.
The genome of NDV is a single-stranded RNA molecule of negative polarity, complementary to the messenger RNA's which code for the virus proteins. The RNA genome is approximately 15,200 nt in size and codes for the following gene products (listed from the 3′ end to the 5′ end of the genomic RNA): nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and large polymerase protein (L) (Chambers et al., 1986).
The RNA is complexed with the NP, P and L proteins to form a ribonucleocapsid particle (RNP) that is surrounded by an envelope that is lined at the inside by the M protein. The envelope contains the F and HN proteins which are involved in attachment and penetration of the host cell.
Replication of NDV is similar to the strategy used by other paramyxovirinae. The initial step is attachment of the virus to the host cell receptors, mediated by the HN protein. Fusion of the viral envelope with the host cell membrane is dependent on the action of both the HN and F proteins and results in the release of the RNP into the cytoplasm where virus replication takes place.
The viral RNA-dependent RNA polymerase (which is part of the RNP) produces complementary transcripts that act as mRNA's and are used by the cell's translation machinery for the synthesis of virus proteins. Due to the accumulation of NP protein, the RNA polymerase complex switches from transcription to replication, resulting in the synthesis of full-length genomic and antigenomic RNA molecules.
Newly formed RNP's are encapsidated at the cellular membrane by the action of the M protein and the F and HN proteins which have accumulated in the cellular plasma membrane. Newly formed virus particles are released from the infected cell by a budding mechanism. For more detailed information about NDV replication see Peeples (1988). For a recent review of the molecular biology of paramyxovirinae see Lamb and Kolakofsky (1996).
Apart from commercial domestic poultry (e.g., chicken, turkey, pheasant, guinea fowl, duck, goose, and pigeon), a wide range of captive, semi-domestic and free-living birds, including migratory waterfowl, is susceptible to NDV and can be primary infection sources (Kaleta and Baldauf, 1988).
The pathogenicity of NDV strains differs greatly with the host. The most resistant species appear to be aquatic birds while the most susceptible are gregarious birds forming temporary or permanent flocks. Chickens are highly susceptible but ducks and geese may be infected and show few or no clinical signs, even with strains which are lethal for chickens.
Newcastle Disease is complicated in that different isolates and strains of the virus may induce enormous variation in the severity of the disease. Beard and Hanson (1984) grouped NDV strains and isolates into different pathotypes that relate to disease signs that may be seen in fully susceptible chickens: 1) viscerotropic velogenic NDV, which produces acute lethal infections in which hemorrhagic lesions are prominent in the gut; and neurotropic velogenic NDV, which produces high mortality preceded by respiratory and neurological signs, but no gut lesions; 2) mesogenic NDV, which produces low mortality, acute respiratory disease and nervous signs in some birds; 3) lentogenic NDV, which produces mild or unapparent respiratory infections or even asymptomatic enteric NDV, avirulent viruses that appear to replicate primarily in the intestinal tract. Some overlap between the signs associated with the different groups has been reported.
The virus enters the body via the respiratory and the intestinal tract or via the eye. In the trachea, the virus is spread by ciliary action and by cell-to-cell spread. After initial multiplication at the introduction site, virus is carried during episodes of viremia to spleen, liver, kidney and lungs. Viruses of some strains reach vital organs like liver and kidney very rapidly so that the birds may die before disease symptoms are overt.
Most viruses reach the central nervous system via the blood before significant amounts of antibody exist. A long, asymptomatic carrier state presumed to occur in psittacines constitutes a potential threat to the poultry industry. A long term carrier state of both lentogenic and velogenic virus may also exist in chickens (Heuschele and Easterday, 1970).
During the replication of NDV it is necessary for the precursor glycoprotein Fo to be cleaved to F1 and F2 for the progeny virus to be infectious (Rott and Klenk, 1988). This posttranslational cleavage is mediated by host cell proteases. If cleavage fails to take place, non-infectious virus particles are produced and viral replication cannot proceed. The Fo protein of virulent viruses can be cleaved by a wide range of proteases, but Fo proteins in viruses of low virulence are restricted in their sensitivity and these viruses can only grow in vivo in certain host cell types and in general cannot be grown in vitro.
Lentogenic viruses only replicate in areas with trypsin-like enzymes such as the respiratory and intestinal tract, whereas virulent viruses can replicate in a range of tissues and organs resulting in fatal systemic infection.
Amino acid sequencing of the Fo precursor has shown that low-virulence viruses have a single arginine (R) that links the F2 and F1 chains, whereas virulent strains possess additional basic amino acids forming two pairs such as K/R-X-K/R-R-F at the site of cleavage. Furthermore, the F2 chain of virulent strains generally starts with a phenylalanine residue whereas that of non-virulent strains generally starts with a leucine.
For a few strains of NDV the HN protein is also produced as a precursor that requires cleavage to be biologically active (Garten et al., 1980; Millar et al., 1988).
Besides cleavability of the F and HN proteins, other viral factors may contribute to pathogenicity. Madansky and Bratt (1978, 1981a, 1981b) have shown that alterations in transcription and translation could modulate growth and cell-to-cell spread of the virus and/or cytopathogenicity.
The initial immune response to infection with NDV is cell mediated and may be detectable as early as 2-3 days after infection with live vaccine strains. This presumably explains the early protection against challenge that has been recorded in vaccinated birds before a measurable antibody response is seen (Gough and Alexander, 1973).
At about 1 week after infection, circulating antibodies may protect the host from re-infection. In the early phase IgM is involved, followed by IgG. Titers and protection peak after about 3 weeks, and gradually decline if without boosting. This means that for older birds, re-vaccinations are necessary.
Only live vaccines administered by the respiratory route stimulate antibody in all mucosal surfaces as well as in serum. Inactivated vaccine, even when applied via the intramuscular route, does not elicit local resistance in the respiratory tract, despite high concentrations of serum antibody.
This stresses the importance of live vaccines capable of presenting viral antigen to the upper respiratory tract to induce both local and systemic immunity. Small droplets penetrate into the lower respiratory tract thereby provoking a mainly humoral immune response, while coarse droplets stimulate local immunity in the upper respiratory tract.
Therefore, aerosols with a wide range of droplet sizes generate the best overall local and humoral immunity.
It should be noted, however, that despite intensive vaccination with current vaccines creating high levels of antibody titers, virus can still be recovered from mucous surfaces.
The identification of Newcastle disease in the USA led to the use of inactivated vaccines (Hofstad, 1953). The observation that some of the enzootic viruses produced only mild disease resulted first in the development of the mesogenic live vaccine Roakin (Beaudette et al., 1949) and, subsequently, in the development of the milder Hitchner B1 (Hitchner and Johnson, 1948) and LaSota (Goldhaft, 1980) strains, which are now the most widely used live vaccines.
NDV live vaccines can be divided into two groups, lentogenic and mesogenic. Mesogenic strains are suitable only for secondary vaccination of birds due to their greater virulence. The immune response increases as the pathogenicity of the live vaccine increases. Therefore, to obtain the desired level of protection without serious reaction, currently vaccination programs are used that involve sequential use of progressively more virulent vaccines, or live vaccines followed by inactivated vaccines.
One of the main advantages of live vaccines is that they may be administered by inexpensive mass application techniques. A common method of application is via drinking water. However, drinking water application must be carefully monitored as the virus may be inactivated by excessive heat and light and by virucidal impurities in the water.
Mass application of live vaccines by sprays and aerosols is also very popular due to the ease with which large numbers of birds can be vaccinated in a short time. It is important to achieve the correct particle size by controlling the conditions under which the particles are generated.
Currently used live vaccines have several disadvantages. The vaccine may still cause disease signs, depending upon environmental conditions and the presence of complicating infections. Therefore, it is important to use extremely mild virus for primary vaccination and, as a result, multiple vaccinations are usually needed. Furthermore, maternally derived antibodies may prevent successful primary vaccination with lentogenic live vaccines.
Inactivated vaccines are usually produced from infectious allantoic fluid which is treated with formalin or beta-propiolactone to kill the virus and mixed with a suitable adjuvant. Inactivated vaccines are administered by injection, either intramuscularly or subcutaneously. Inactivated vaccines are expensive to produce and to apply.
However, inactivated oil-emulsion vaccines are not as adversely affected by maternal immunity as live vaccines and they can be used in day-old chicks. Advantages of inactivated vaccines are the low level of adverse reactions in vaccinated birds, the high level of protective antibodies, and the long duration of protection. None of the above vaccines can serologically be differentiated from wild-type NDV.
The development of recombinant viral vaccines has been of interest to the poultry industry for a number of years. The concept is to insert genes of critical immunizing epitopes of a disease agent of interest into a nonessential gene of a vector virus. Vaccination with the recombinant virus thus results in immunization against both the vector virus as well as the disease agent of interest.
Several types of viruses have been evaluated as potential live viral vaccines for poultry. Two avian viruses that have received most attention are fowl pox virus (FPV) and herpes virus of turkeys (HVT). Fowl pox virus is a DNA virus that has a large genome and hence is considered to have ample room to carry foreign genes.
When attenuated, FPV does not cause clinical disease and is commonly used as a vaccine in chickens. HVT is also a DNA virus and is classified as serotype III of the Marek's disease virus (MDV) family. HVT is non-pathogenic for chickens yet cross-protective against MDV and is commonly used to vaccinate chickens against Marek's disease.
It has been shown that protection against Newcastle disease can be induced by using recombinant HVT or FPV vaccines (Morgan et al., 1992, 1993; Heckert et al., 1996; Boursnell et al., 1990; Taylor et al., 1990).
However, the onset of protection against Newcastle disease following vaccination with such recombinant vaccines that express either the NDV F protein or both the F and HN proteins was severely delayed compared to that following vaccination with a conventional live or inactivated NDV vaccine, possibly because the recombinant vaccines do not provide a wide enough immunological specter of antigenically relevant NDV epitopes other than those found on the NDV protein that is expressed by the recombinant vaccine or are not properly presented to the immune system.
Furthermore, local (mucosal, respiratory or enteric) protection was not effectively induced in birds vaccinated with the recombinants. This is a serious drawback since vaccines used for primary vaccination against respiratory diseases must induce local immunity to prevent infection and spread of virulent viruses that infect chickens reared under field conditions.
Antibodies against NDV which are capable of protecting the host can be measured in virus neutralization tests. However, since the neutralization response appears to parallel the hemagglutination-inhibition (HI) response, the latter test is frequently used to assess the protective response, especially after vaccination.
Antibodies against both the F and HN proteins can neutralize NDV. However, antibodies against the F protein appear to induce greater neutralization than those directed against HN in in vivo and in vitro tests (Meulemans et al., 1986).
The presence of specific antibodies to NDV in the serum of a bird gives little information on the infecting strain of NDV and therefore has limited diagnostic value.
The omnipresence of lentogenic NDV strains in birds in most countries and the almost universal use of live vaccines that cannot be distinguished, at least not serologically from wild-type NDV, mean that the mere demonstration of infection is rarely adequate cause for control measures to be imposed. Since field disease may be an unreliable measure of the true virulence of the virus, it is necessary to further characterize the virus that is found.
At present, the only method of Newcastle disease diagnosis which allows characterization of the infecting strain is virus isolation followed by pathogenicity testing. At present, three in vivo tests are used for this purpose: 1) mean death time (MDT) in eggs; 2) intracerebral pathogenicity index (ICPI) in one-day-old chickens; 3) Intravenous pathogenicity index (WVPI) in 6-week-old birds.
These tests suffer from a number of drawbacks, such as the availability of animals, poor reproducibility, and the relatively long duration of the tests. Last but not least, these tests do not allow a simple serological identification of poultry vaccinated with a vaccine or infected with a wild-type strain.
As an alternative to in vivo tests, the polymerase chain reaction (PCR) has been successfully used to distinguish between virulent and non-virulent and non-virulent isolates (Stauber et al., 1995; Kant et al., 1997), however, again serological differentiation is not possible.
The raising of poultry and trade of their products is now organized on an international basis, frequently under management of multinational companies. The threat of Newcastle disease has proven a great restraint on such trade.
Successful control of Newcastle disease will only be approached when all countries report outbreaks. However, international agreements are not simple due to enormous variation in the extent of disease surveillance in different countries. Some countries do not vaccinate and would not want any form of NDV introduced in domestic poultry because vaccinated poultry cannot be distinguished from those infected with wild-type NDV.
Others only allow the use of specific live vaccines and consider other vaccines as unacceptably virulent. Yet other countries have the continued presence of circulating highly virulent virus, which is not recognized as such because overt disease is masked by vaccination.
In many countries legislation exists to control Newcastle disease outbreaks that may occur. National control measures are directed at prevention of introduction and spread. Most countries have restrictions on trade in poultry products, eggs, and live poultry. Most countries have established quarantine procedures for importation, especially for psittacine birds.
Some countries have adopted eradication policies with compulsory slaughter of infected birds, their contacts, and products. Others require prophylactic vaccination of birds even in the absence of outbreaks, while some have a policy of ring vaccination around outbreaks to establish a buffer zone.
A need exists for better vaccines and for better diagnostic methods which can be used to control Newcastle disease. Due both to large differences in the dose that is received by individual birds during mass application of live vaccines and to variation in levels of maternal immunity in young chickens, post-vaccination reactions with live vaccines are inevitable. This is one of the main concerns of farmers in countries where vaccination is compulsory.
Furthermore, many vaccines are mixtures of sub-populations. When cloned, these sub-populations may differ significantly from each other in immunogenicity and pathogenicity (Hanson, 1988).
However, the largest drawback of currently used live vaccines and inactivated vaccines is the fact that vaccinated animals cannot be distinguished from infected animals with currently used screening techniques such as hemagglutination-inhibition or virus neutralization tests.
Virulent field-virus may still spread in vaccinated flocks since disease symptoms are masked by vaccination. Since virus isolation and characterization of virulence by in vivo techniques is not feasible on a large scale, there is a great need for new and effective attenuated live vaccines which can be serologically discriminated from field-viruses.
Such vaccines, called NDV marker vaccines (and accompanying diagnostic methods and kits) which should provide the fullest possible immunological specter of antigenically relevant NDV epitopes, and yet should be serologically distinct from wild-type NDV are not yet available.